新型肾癌循环肿瘤细胞的检测及单细胞分析的研究

The kidney cancer circulating tumor cells (CTC) and the metastasis of kidney cancer is closely related. The detection of CTC will enhance our understanding of the process of tumor metastasis. But there is no method can completely eliminate the high false-positive and false-negative.This study will base on the latest progress of tumor-specific replication-defective adenovirus and single cell analysis,build carbonic anhydrase IX (CAIX) tumor-specific promoter which controlled GFP reporter adenovirus replication.The replication necessary structure of the adenovirus E1A will be controlled by CAIX promoter.E3 structure will be replaced with GFP structure.This adenovirus will replicate only within the renal cell carcinoma tumor cells but not in normal cells. CD45 staining exclusion will eliminates leukocyte effect.The individual GFP+/CD45- cell will be counted and isolated using an inverted micromanipulator.Combined with the current latest single cell analysis techniques to anatomize the CTC and compare with normal kidney tissue in the genetic level, this study will develop an entirely new CTC detection and analysis techniques to minimize false-negtive and false-positive. It will provide new ideas for the accurate detection of CTC and new method to analyse CTC. This study is great significant to early detection metastasis invasion of kidney cancer, cancer treatment monitoring and prognosis assessment.

肾癌循环肿瘤细胞（CTC）与肾癌的转移密切相关，CTC的检测增进了我们对于恶性肿瘤转移过程的理解。但目前尚无一种方法可完全解决假阳性或假阴性较高的问题。本研究将根据当前肿瘤特异性复制缺陷型腺病毒和单细胞分析的最新进展，构建碳酸酐酶IX（CAIX）启动子控制的肿瘤特异性GFP报告基因腺病毒。该腺病毒的复制必需结构E1A受控于CAIX启动子，并用GFP取代E3结构，使得该腺病毒仅仅能够在肾癌肿瘤细胞内复制而不能在正常细胞中复制。并应用CD45染色排除技术消除白细胞影响,计数并显微操作获取GFP+/CD45-细胞。结合当前最先进的单细胞分析技术，从基因水平检测CTC与正常肾组织的差异。本研究如获得成功，将开发出一种全新的CTC检测及分析技术，最大限度降低假阴性和假阳性。为准确检测CTC并予分析，为CTC检测提供新思路。本研究对早期发现肾癌的恶性侵袭、肿瘤的治疗监测、患者的预后评估等具有重要意义。

肾癌(RCC)，又称为肾细胞癌、肾腺癌，起源于泌尿小管上皮。统计表明，RCC确诊时已有30%—40%发生远处转移]。循环肿瘤细胞定植在器官就造成了肿瘤的播散转移——这种观点已经得到全世界肿瘤学家的广泛承认。血液中检测到循环肿瘤细胞(Circulating Tumor Cells, CTC)并不意味着有远处转移，但发生转移的概率将大大增加。根据分离得到的CTC进行生物学分析，对进入CTC发生远处种种植的可能性进行预判，将对肾癌的诊断、治疗、预后判断具有重大的意义。本课题在国家自然科学基金的资助下，成功构建了肾细胞癌肿瘤特异性启动子CAIX。通过分子生物学实验技术，成功构建了包含肾癌启动子的Rp-CAIX-E1A作为穿梭质粒。T-IRES-HrGFP作为报告基因（1301bp）、对穿梭治疗进行改造为Rp-CAIX-E1A-IRES-HrGFP，使其具有了绿色荧光报告基因及IRES强启动子。应用同源重组技术成功构建重组质粒AdEasy-1- CAIX-E1A- IRES-HrGFP。应用脂质体病毒包装系统，在293包装细胞中，完成第5型肾癌组织特异性报告基因腺病毒Ad5- CAIX-E1A- IRES-HrGFP的包装过程。通过病毒操作技术，完成了腺病毒的扩增、纯化、鉴定和滴度测定。应用人白血病细胞 K562作为白细胞背景，检测了少量混入的肾癌细胞ACHN,A-498, 786-0，检测到了确切的荧光信号。实验达到了预期效果。